Vitamin D3 analog useful for the treatment of osteoporosis

ABSTRACT

The present invention relates to the vitamin D 3  analog (1R,3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol which is useful in the treatment of osteoporosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under Title 35, United States Code 119(e) from Provisional Application Ser. No. 60/633,670, filed Dec. 6, 2004, which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the vitamin D₃ analog (1R,3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethyli-dene}-cyclohexane-1,3-diol which is useful in the treatment of bone disorders, more in particular -osteoporosis. The present invention further relates to compositions, more in particular pharmaceutical compositions comprising said vitamin D₃ analog, to a method of treating humans with said analog, and to a process for preparing said analog.

DESCRIPTION OF THE DRAWING

FIG. 1 shows the effect on bone (A) and serum calcium (B) levels of ovariectomized Spargue-Dawley rats upon treatment with the compound of the present invention, (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol at different dosages.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the vitamin D₃ analog (1R,3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol having the formula:

For the purposes of the present invention, in order to describe in detail aspects of the vitamin D₃ analog disclosed herein, the following numbering system is used when referring to the carbon atoms which comprise said analog.

The following stereochemistry has been assigned to carbons 1, 3, 13, and 21 of said analog:

The analog of the present invention comprises an upper CF-Ring scaffold connected to a lower A-Ring scaffold as depicted herein below.

The upper CF-Ring scaffold having the (R,R) stereochemistry at carbons 13 and 21 is prepared via key intermediates 10 and 14a. Once formed the CF-Ring scaffold is coupled with the lower A-Ring scaffold.

The following is a detailed description of a process for preparing (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol. The present invention also relates to a number of intermediates, most of them having a specific stereochemistry, and which are useful in the preparation of the final vitamin D₃ analog of this invention. The preparation and the detailed formulae of a first set of these intermediates are shown in the following scheme I comprising a sequence of eleven process steps for making pure isomers of 1,4-dioxa-dispiro[4.1.5.3]pentadec-8-ene. Each step will now be illustrated in details, based on specific starting compounds, reactive agents, catalysts, solvents, temperature ranges and the-like, but the skilled person will readily understand that the specific materials and conditions disclosed herein may be replaced with similar or equivalent materials and conditions without significantly altering the resulting product of the relevant step, except maybe for the reaction yield of such step.

Scheme I

Synthesis of a pure isomer of 1,4-dioxa-dispiro[4.1.5.3]pentadec-8-ene starts, as a first step (a), with the halogenation, preferably the bromination, of 3-ethoxy-cyclohex-2-enone, e.g. according to the procedure disclosed by Hara et al. in J. Am. Chem. Soc. (1 999) 121, 3072-3082:

(preferred reagents and conditions for step (a): N-bromosuccinimide as a reagent, CH₂Cl₂ as a solvent; temperature range from about 0° C. to about 40° C.).

In a second step (b), the 2-bromo-3-ethoxy-cyclohex-2-enone resulting from step (a) is n-butylated for instance, but without limitation, by means of 1-chloro-4-methoxybutane (the latter may be prepared according to the procedures disclosed by Hara et al. in J. Org. Chem. (1975) 40, 2786-2791 and by De Buyck et al. in Bull. Soc. Chim. Belg. (1992) 101, 807-815) in the presence of an effective amount of an organometallic derivative such as a Grignard reagent. In a more specific embodiment in order to improve butylation yield, said step may additionally be performed in the presence of a stoechiometric amount of cerium trichloride. This specific embodiment is believed to result in the formation of an organocerium active species as shown in the scheme below, although said presumed active species was not isolated:

(preferred reagents and conditions for step (b): THF as a solvent; temperature range from about 0° C. to about 40° C. after refluxing to create the Grignard reagent; magnesium, 1-chloro-4-methoxybutane and cerium trichloride as active reagents).

In a third step (c), the 2-bromo-3-(4-methoxybutyl)-cyclohex-2-enone resulting from step (b) is stereoselectively reduced by means of an effective amount of a reducing agent in the presence of a catalytic amount of a suitable stereoselective reduction catalyst. Suitable reducing agents for this reduction step include, but are not limited to, borane, catecholborane and borohydride reagents. Suitable stereoselective reduction catalyst for this reduction step include, but are not limited to, substantially pure methyloxazaborolidinone isomers and metal complexes, either monometallic, homobimetallic or heterobimetallic, such as lithium aluminum hydrides or transition metal complexes, having one or more chiral ligand. The skilled person understands that this step is very; important for the present invention since it determines the success of the whole synthetic route up to the desired following stereoisomeric intermediates and the desired vitamin D₃ analog of the invention. Therefore the skilled person, based on the general knowledge relating to reducing catalysts and agents, will make a careful selection of the reaction conditions in view of various considerations such as reaction yield, productivity and, mainly, enantiomeric excess of one stereoisomer of the resulting product with respect to the other stereoisomer.

(preferred reagents and conditions for step (c) as shown above: (R)-methyloxazaborolidinone as a catalyst, catecholborane as a reducing agent, toluene as a solvent; temperature range from about −95° C. to about 25° C., more preferably from −78° C. to about 0° C).

In a fourth step (d), the 2-bromo-3-(4-methoxybutyl)-cyclohex-2-enol stereoisomer resulting from step (c) is submitted to a 3,3-sigmatropic rearrangement by means of a dimethylamino-dimethylacetal (preferably in an at least stoechiometric amount), preferably in the presence of a suitable solvent:

(preferred reagents and: conditions for step (d) include: (CH₃)₂N[CCH₃(OCH₃)₂] as a reagent, toluene as a solvent; temperature: reflux of the solvent).

In a fifth step (e), the 2-[2-bromo-1-(4-methoxybutyl)-cyclohex-2-enyl]-N,N-dimethylacetamide stereoisomer resulting from step (d) is reduced by means of a reducing agent such as a metal hydride, preferably in the presence of a suitable solvent and optionally in the presence of a free radical initiator:

(preferred reagents and conditions for step (e): BU₃SnH as the reducing agent, azobis-isobutyronitrile as a free radical initiator; THF a solvent; temperature: reflux of the solvent).

In a sixth step (f), the 2-[1-(4-methoxybutyl)-cyclohex-2-enyl]-N,N-dimethylacetamide stereoisomer resulting from step (e) is submitted to ether cleavage, preferably in the presence of a Lewis acid reagent, a nucleophile reagent and an effective amount of suitable catalyst such as a crown ether.

(preferred reagents and conditions for step (f) include: BBr₃ as a Lewis acid, Nal as a nucleophilic reagent, and 15-crown-5 as a catalyst, CH₂Cl₂ as a solvent; temperature range from about −40° C. to about −20° C.).

In a seventh step (g), the 2-[1-(4-hydroxybutyl)-cyclohex-2-enyl]-N,N-dimethylacetamide stereoisomer resulting from step (f) is oxidized in the presence of a suitable oxidation reagent, preferably a chromium (VI) compound, preferably at moderate temperature and in the presence of a suitable solvent:

(preferred reagents and conditions for step (g) include: pyridinium dichromate as an oxidation reagent, DMF as a solvent; temperature range from about 10° C. to about 40° C.).

In an eighth step (h), the 4-(1-dimethylcarbamoylmethyl-cyclohex-2-enyl)-butyric acid stereoisomer resulting from step (g) is esterified, preferably in two sub-steps including first a strong base at elevated temperatures, and secondly the presence of an aliphatic alcohol optionally in the presence of an effective amount of an esterification catalyst:

(preferred reagents and conditions for step (h) include: first. (i) KOH, dioxane/water mixture as a solvent, temperature range from about 160° C. to about 240° C.; then (ii) diazomethane (CH₂N₂), MeOH).

In a ninth step (i), the 4-(1-dimethylcarbamoylmethyl-cyclohex-2-enyl)-butyric acid methyl ester stereoisomer resulting from step (h) is submitted to a Dieckmann condensation into a β-keto ester preferably in the presence of a strong base such as a sodium alkoxide or a lithium amide, and optionally in the presence of a suitable solvent:

(preferred reagents and conditions for step (i) include: lithium diisopropylamide as a base, THF as a solvent; temperature range from about −80° C. to about 40° C.).

In a tenth step (1), the 2-hydroxy-spiro[5.5]undecan-2,7-diene-3-carboxylic acid methyl ester stereoisomer resulting from step (i) is submitted to oxidative decarboxylation, preferably in the presence of an oxidative agent:

(preferred reagents and conditions for step (1) include: NaCl, water, and DMSO both as a solvent and an oxidative agent; temperature range from about 140° C. to about 180° C.).

In an eleventh step (k); the spiro[5.5]undec-7-en-2-one stereoisomer resulting from step (i) is submitted to ketalization by means of an aliphatic diol, optionally in the presence of a suitable acidic catalyst:

(preferred reagents and conditions for step (k) include: HOCH₂CH₂OH as a reagent, p-toluenesulfonic acid as a catalyst, toluene as a solvent; temperature at reflux of the solvent).

EXAMPLE 1 Preparation of 1,4-dioxa-dispiro[4.1.5.3]pentadec-8-ene (11)

The preparation of 1,4-dioxa-dispiro[4.1.5.3]pentadec-8-ene was effected via the principles of the eleven steps synthetic route described above, and more specifically as outlined hereunder.

a) Preparation of 2-bromo-3-ethoxy-cyclohex-2-enone (1)

To a solution of 3-ethoxy-cyclohex-2-enone (70 g, 499 mmol) in CH₂Cl₂ (300 mL) as a solvent at 0° C. under argon atmosphere was added in portions N-bromosuccinimide (91 g, 511 mmol) over a period of 1 hour. The suspension was stirred at 0° C. for 1 hour and then at room temperature for 2 hours. The solvent was removed in vacuo and CH₂Cl₂ was added. The solution was washed twice with a cold saturated NaHCO₃ solution and with cold H₂O, then dried over anhydrous MgSO₄, the solvent removed in vacuo and the resulting solid residue was triturated several times with diethylether (Et₂O), the resulting solid dried in vacuo to afford 101 g. (92% yield) of the desired product as white crystals which were characterized as follows:

R_(f) (Et₂O) 0.33;

ultraviolet (UV) absorption band (MeOH) at λ_(max) 273 nm;

infrared (IR) main absorption bands (KBr) ν at 2966, 2939, 1657, 1568, 1430, 1397, 1371, 1348, 1324, 1295, 1260, 1246, 1195, 1150, 1107, 1076, 1030, 959, 922, 850, 798, 614 and 470 cm⁻¹;

proton nuclear magnetic resonance (¹H NMR) (500 MHz, CDCl₃): δ 4.21 (2 H, q, J=7.0 Hz), 2.69 (3 H, t, J=6.2 Hz), 2.55-2.52 (2 H, m), 2.07-2.02 (2 H, m), 1.43 (2 H, t, J=7.0 Hz) ppm;

carbon nuclear magnetic resonance (¹³C NMR) (50 MHz, CDCl₃): δ 191.0 (C), 172.8 (C), 103.0 (C), 65.2 (CH₂), 36.7 (CH₂),.27.2 (CH₂), 20.6 (CH₂) and 15.1 (CH₃) ppm; and

mass spectrum (MS) m/z (%): 220/218 (M⁺, 16/20), 192/190 (25/25), 164/162 (75/100), 149 (12), 111 (15), 67 (21) and 55 (51).

b) Preparation of 2-bromo-3-(4-methoxy-butyl)-cyclohex-2-enone (2)

To magnesium turnings (9.84 g, 405 mmol) in dry tetrahydrofuran (THF) (42 mL) as a solvent were added a few iodine crystals and a few drops of CH₂Br₂. A solution of 1-chloro-4-methoxybutane.(46.8 g, 382 mmole) in dry THF (90 mL) was added dropwise at a rate sufficient to maintain gentle reflux. The reaction mixture was then refluxed for 1 hour and then cooled to room temperature. The resulting Grignard reagent was then added dropwise to a suspension of CeCl3 (94.2 g, 382 mmol) in dry THF (750 mL) at 0° C. After stirring at room temperature for 2 hours, a solution of 2-bromo-3-ethoxy-cyclohex2-enone (1) (60 g, 274 mmol) in dry THF (300 mL) was added dropwise at 0° C. The reaction mixture was stirred an additional 2 hours at room temperature and then quenched by adding a saturated NH₄Cl solution (1000 mL). The mixture was further acidified to approximately pH 1 by the addition of a 5% HCl solution and stirring was continued for an additional 2 hours. The organic layer was separated, the aqueous layer extracted with Et₂O and the combined organic layers were dried over anhydrous MgSO₄. The solvent was removed in vacuo and the residue purified over silica (using a isooctane/EtOAc 7:3 mixture as an eluent) to afford 65.5 g (92% yield) of the desired product as a light-yellow oil which was characterized as follows:

R_(f) (isooctane/ethyl acetate [EtOAc], 7:3) 0.30;

UV (MeOH) λ_(max) 254 nm;

IR (KBr film) ν 2929, 2865, 2828, 1682, 1595, 1455, 1427, 1385, 1337, 1313,1272, 1175, 1117, 982 and 795 cm⁻¹;

¹H NMR (500 MHz, CDCl₃): δ 3.38 (2 H, t, J=5.9 Hz), 3.31 (3 H, s), 2.56-2.53 (2 H, m), 2.50-2.47 (4 H, m), 1.99-1.94 (2 H, m), 1.65-1.56 (4 H, m) ppm;

¹³C NMR (50 MHz, CDCl₃): δ 191.2 (C), 163.7 (C), 122.6 (C), 72.1 (CH₂), 58.6 (CH₃), 38.9 (CH₂), 37.8 (CH₂), 32.3 (CH₂), 29.4 (CH₂), 23.5 (CH₂) and 21.9 (CH₂) ppm;

MS m/z (%) 230/228 (M⁺-MeOH, <1/1), 200/188 (4/4), 181 (3), 149 (20), 131 (7), 121 (12), 107 (16), 93 (29), 91 (24), 79 (60), 77 (40), 65 (20), 51 (29) and 45 (100).

c) Preparation of 2-bromo-3-(4-methoxybutyl)-cyclohex-2-enol (3)

To a solution of 2-bromo-3-(4-methoxy-butyl)-cyclohex-2-enone (2) (32.0 g, 123 mmol) in dry toluene (480 mL) at −95° C. was added (R)-methyl-oxazaborolidinone (1 M solution in toluene; 25 mL, 25 mmol). Catecholborane (1 M solution in toluene; 160 mL, 160 mmol) was added dropwise at −95° C. over a period of 10 hours and the reaction mixture stirred at ˜78° C. overnight. A 1 M NaOH solution (500 mL) was added and temperature was allowed to rise to room temperature. The organic layer was separated and the aqueous layer extracted with. Et₂O. The combined organic layers were washed with a 1 M NaOH solution and dried over anhydrous MgSO₄. The solvent was removed in vacuo, and the resulting residue purified over silica (isooctane/EtOAc, 8:2) to afford 31.5 g (97% yield) of the desired product as a colorless oil which was characterized as follows:

R_(f) (n-pentane/Et₂O, 7:3) 0.33;

optical rotation at room temperature: [α]_(D) ^(rt) −70.0 (c=1.13, CHCl₃);

IR (KBr film) ν 3418, 2935, 2864, 2828, 1645, 1455, 1387, 1337, 1264, 1165, 1118, 1078, 990, 970, 939, 813 and 733 cm⁻¹;

¹H NMR (500 MHz, CDCl₃): δ 3.38 (2 H, t, J=6.4 Hz), 3.32 (3 H, s), 2.38 (1 H, d, J=3.8 Hz), 2.20 (2 H; dd, J=7.8, 7.8 Hz), 2.14 (2 H, ABt, J=17.3, 5.0 Hz), 2.07 (1 H, ABdd, J=17.3, 8.3, 5.6 Hz, 1.87-1.83 (2 H, m), 1.79-1.71 (1 H, m), 1.65-1.55 (3 H, m) and 1.51-1.45 (2 H, m) ppm;

¹³C NMR (50 MHz, CDCl₃): δ 140.5 (C), 122.9 (C), 72.5 (CH₂), 71.0 (CH), 58.5 (CH₃), 36.9 (CH₂), 32.0 (CH₂), 31.3 (CH₂), 29.3 (CH₂), 23.6 (CH₂), 18.3 (CH₂) ppm;

MS m/z (%) 246/244 (M⁺-H₂O, <1/1), 214/212 (3/3), 165 (10), 133 (42), 123 (19), 105 (25), 91 (77), 79 (38), 67 (26), 55 (28), 53 (26), 45 (100) and 41 (33).

elemental analysis: calculated for C₁₁H₁₉BrO₂: C, 50.20; H, 7.28; found: C, 50.17; H, 7.17.

d) Preparation of 2-[2-bromo-1-(4-methoxybutyl)-cyclohexy-2-enyl]-N,N-dimethyl-acetamide (4)

To a solution of 2-bromo-3-(4-methoxybutyl)-cyclohex-2-enol isomer (3) (24.5 g, 93.1 mmol) in dry toluene (400 mL) was added N,N-dimethylacetamide dimethyl acetal (27.2 mL, 186.2 mmol). The reaction mixture was brought to reflux for 8 hours removing the generated MeOH by azeotropic distillation. The solvent was removed in vacuo and the residue purified over silica (using cyclohexane/EtOAc 1:1 as the eluent) to afford 29.4 g (95% yield) of the desired product as a light-yellow oil which was characterized as follows:

R_(f) (isooctane/EtOAc, 1:1) 0.28;

optical rotation [α]_(D) ^(rt) −41.4 (c=1.67, CHCl₃);

IR (KBr film): ν 2934, 2865, 1644, 1490, 1456, 1393, 1258, 1179, 1149, 1118, 968, 891, 861, 813 and 761 cm⁻¹.

¹H NMR (500 MHz, CDCl₃): δ 6.13 (1 H, dd, J=4.2, 4.2 Hz), 3.38 (1 H, ABt, J=9.3, 6.5 Hz), 3.36 (1 H, ABt, J=9.3, 6.6 Hz), 3.31 (3 H, s), 3.06 (3 H, s), 2.92 (3 H, s), 2.59 (1 H, AB, J=14.9 Hz), 2.44 (1 H, AB, J=14.9 Hz), 2.32 (1 H, m), 2.09-1.98 (2 H, m), 1.77-1.71 (1 H, m), 1.67-1.52 (6 H, m) and 1.39-1.26 (2 H, m) ppm;

¹³C NMR (125 MHz, CDCl₃): δ 170.7 (C), 133.0 (C), 132.0 (CH), 72.6 (CH₂), 58.4 (CH₃), 43.8(C), 39.7 (CH₂), 38.2 (CH₂), 38.0 (CH₃), 35.4 (CH₃), 30.7 (CH₂), 30.0 (CH₂), 27.8 (CH₂), 20.5 (CH₂), 18.6 (CH₂) ppm; MS m/z (%) 252 (M⁺-Br, 3), 91 (8), 87 (3), 72 (14) and 45 (100);

elemental analysis: calculated for C₁₅H₂₆BrNO₂: C, 54.22; H, 7.89; N, 4.22; found: C, 54.04; H, 8.05; N, 4.22.

e) Preparation of 2-[1-(4-methoxybutyl)-cyclohex-2-enyl]-N,N-dimethyl-acetamide (5)

To a solution of 2-[2-bromo-1-(4-methoxybutyl)-cyclohexy-2-enyl]-N,N-dimethyl-acetamide isomer (4) (37.0 g, 111 mmol) in THF (600 mL) was added tributyltin hydride (n-Bu₃SnH) (35.3 mL, 133 mmol) followed by azobisisobutyronitrile (1.8 g, 11.1 mmol). The reaction mixture was brought to reflux for 4 hours and the solvent removed in vacuo. The resulting residue was purified over silica (gradient elution: cyclohexane/EtOAc, 6:4 to EtOAc 100%) to afford 25.9 g (92% yield) of the desired product as a light-yellow oil which was characterized as follows:

R_(f) (isooctane/EtOAc, 1:1) 0.24;

optical rotation: [α]_(D) ^(rt) −24.0 (c=1.59, CHCl₃);

IR (KBr film): ν 3013, 2931, 2864, 1637, 1490, 1449, 1384, 1261, 1114, 1061, 955 and 732 cm⁻¹;

¹H NMR (500 MHz, CDCl₃) δ 5.62 (1 H, ABt, J=10.2, 3.7 Hz), 5.50 (1 H, AB, J=10.2 Hz), 3.32 (2 H, t, J=6.6 Hz), 3.26 (3 H, s), 2.96 (3 H, s), 2.87 (3 H, s), 2.40 (1 H, AB, J=14.2 Hz), 2.19 (1 H, AB, J=14.2 Hz), 1.93-1.84 (2 H, m), 1.63-1.43 (8 H, m) and 1.32-1.20 (2 H, m) ppm;

¹³C NMR (50 MHz, CDCl₃) δ 171.3 (C), 134.3 (CH), 126.3 (CH), 72.6 (CH₂), 58.2 (CH₃), 40.9 (CH₂), 39.5 (CH₂), 38.1 (CH₃), 37.1 (C), 35.1 (CH₃), 32.5 (CH₂), 30.0 (CH₂), 24.7 (CH₂), 20.3 (CH₂) and 18.8 (CH₂) ppm;

MS m/z (%) 253 (M⁺, 5), 238 (10), 210 (2), 166 (12), 87 (59), 72 (47) and 45 (100);

Elemental analysis: calculated for C₁₅H₂₇NO₂: C, 71.10; H, 10.74; N, 5.53; found: C, 70.97; H, 10.91; N, 5.45.

f) Preparation of 2-[1-(4-hydroxybutyl)-cyclohex-2-enyl-N,N-dimethyl-acetamide (6)

To a solution of 2-[1-(4-methoxybutyl)-cyclohex-2-enyl]-N,N-dimethyl-acetamide isomer (5) (51.0 g, 201 mmol), Nal (60 g, 400 mmol) and 15-crown-5 (55 g, 250 mmol) in CH₂Cl₂ (1000 mL) at −30 ° C. was added dropwise BBr₃ (1 M solution in CH₂Cl₂; 241 mL, 241 mmol). The reaction mixture was stirred at −30 ° C. for 1 hour then a saturated NaHCO₃ solution (1000 mL) was added. The organic layer was separated and the aqueous layer extracted with CH₂Cl₂. The combined organic layers were washed with a saturated Na₂SO₃ solution and dried over anhydrous MgSO₄. The solvent was removed in vacuo and the residue purified over silica (gradient elution: Et₂O 100% to Et₂O/MeOH, 95:5) to afford 39.9 g (83% yield) of the desired product as a viscous oil which was characterized as follows:

R_(f) (EtOAc) 0.25;

optical rotation [α]_(D) ^(rt) −21.4 (c=1.03, CHCl₃);

IR (KBr film): ν 3409, 3012, 2932, 2863, 1626, 1498, 1457, 1398, 1261, 1146, 1060, 923 and 730 cm⁻¹;

¹H NMR (500 MHz, CDCl₃): δ 5.65 (1 H, ABt, J=10.2, 3.7 Hz), 5.48 (1 H, AB, J=10.2 Hz), 3.68-3.59 (2 H, m), 3.00 (3 H, s), 2.91 (3 H, s), 2.79 (1 H, br s), 2.47 (1 H, AB, J=13.9 Hz), 2.24 (1 H. AB, J=13.9 Hz), 1.98-1.89 (2 H, m) and 1.67-1.31 (10 H, m) ppm;

¹³C NMR (50 MHz, CDCl₃) δ 171.8 (C), 134.6 (CH), 126.8 (CH), 61.5 (CH₂), 40.3 (CH₂), 38.5 (CH₃), 38.3 (CH₂), 37.4 (C), 35.5 (CH₃), 35.4 (CH₂), 32.4 (CH₂), 25.0 (CH₂), 19.2 (CH₂) and 18.9 (CH₂) ppm;

MS m/z (%) 239 (M⁺, 3), 196 (2), 166.(12), 121 (2), 87 (100), 79 (35), 72 (90) and 45 (25); and

elemental analysis: calculated for C₁₄H₂₅NO₂: C, 70.25; H, 10.53; N, 5.85; found: C, 70.10; H, 10.49; N, 5.71.

g) Preparation of 4-(1-dimethylcarbamoylmethyl-cyclohex-2-enyl)-butyric acid

To a solution of 2-[1-(4-hydroxybutyl)-cyclohex-2-enyl]-N,N-dimethylacetamide (6) (13.7 g, 57.1 mmol) in DMF (200 mL) was added pyridinium dichromate (65.3 g, 241.0 mmol) and the mixture stirred at room temperature for 24 hours. The reaction mixture was poured into H₂O (200 ML) and extracted five times with ethyl acetate. The combined organic layers were washed with a saturated NaCl solution and dried over anhydrous MgSO₄. The solvent was removed in vacuo, the residual DMF was removed by vacuum distillation (Kugelrohr-type) to afford 13.3 g of the desired product which was used without further purification.

h) Preparation of 4-(1-methoxycarbonylmethyl-cyclohex-2-enyl)-butyric acid methyl ester (8)

The 4-(1-dimethylcarbamoylmethyl-cyclohex-2-enyl)-butyric acid isomer (7) from the previous step (13.3 g, 52.0 mmol; viscous oil) was dissolved in 1,4-dioxane (75 mL), KOH (1 M solution in H₂O; 225 mL) was added and the reaction mixture was heated at 200 ° C. (autoclave) for 4 hours. The reaction mixture was allowed to cool and then acidified using a 37% HCl solution. The aqueous layer was then repeatedly extracted with CH₂Cl₂. The combined organic layers were dried over anhydrous MgSO₄ and the solvent removed in vacuo. The resulting residue was dissolved in MeOH (200 mL) and a solution of CH₂N₂ in Et₂O added at 0° C. with vigorous stirring until a yellow color persists. Excess CH₂N₂ was destroyed by the addition of silica gel, the mixture filtered, and the filtrate dried-over anhydrous MgSO₄. The solvent was removed in vacuo and the resulting residue purified over silica (isooctane/EtOAc, 9:1) to afford 11.3 g (77% yield over 2 steps) of the desired product as a colorless oil which was characterized as follows:

R_(f) (n-pentane/Et₂O, 8:2) 0.42;

[α]_(D) ^(rt) −1.7 (c=1.25, CHCl₃;

IR (KBr film): ν 3011, 2933, 2867, 1737, 1436, 1356, 1256, 1194, 1163, 1096, 1014, 882 and 732 cm⁻¹;

¹H NMR (500 MHz, CDCl₃): δ 5.70 (1 H, ABt, J=10.2, 3.7 Hz), 5.49 (1 H, ABt, J=10.2, 1.9 Hz), 3.66 (3 H, s), 3.64 (3 H, s), 2.34 (1 H, AB, J=13.6 Hz), 2.31 (1 H, AB, J=13.6 Hz), 2.28 (2 H, t, J=7.5 Hz), 2.01-1.88 (2 H, m) and 1.65-1.38.(8 H, m) ppm;

¹³C NMR (50 MHz, CDCl₃): δ 174.1 (C), 172.3 (C), 133.4 (CH), 127.5 (CH), 51.5 (CH₃), 51.3 (CH₃), 43.9 (CH₂), 39.2 (CH₂), 36.8 (C), 34.5 (CH₂), 32.3 (CH₂), 24.8 (CH₂), 19.5 (CH₂) and 18.8 (CH₂) ppm;

MS m/z (%) 254 (M⁺, <1), 223(5), 222 (9), 190 (5),.181 (26), 180 (42), 153 (37), 149 (44), 121 (43), 107 (44), 93 (97), 79 (100), 59 (64) and 42 (35); and

elemental analysis: calculated for C₁₄H₂₂O₄: C, 66.12; H. 8.72; found:: C, 66.24; H, 8.79.

(i) Preparation of 2-hydroxy-spiro[5.5undecan-2,7-diene-3-carboxylic acid methyl ester (9)

To a solution of the 4-(1-methoxycarbonylmethyl-cyclohex-2-enyl)-butyric acid methyl ester isomer (8) (31.0 g, 122 mmol) in dry THF (580 mL) at −78° C. was added dropwise lithium diisopropylamide (2 M solution in n-heptane; 122 mL, 244 mmole). The reaction mixture was allowed to warm to room temperature and stirred for 2 hours. A saturated NH₄Cl solution (700 mL) was added and the aqueous layer repeatedly extracted with Et₂O. The combined organic layers were dried over anhydrous MgSO₄ and the solvents removed in vacuo. The resulting residue was purified over silica (n-pentane/Et₂O, 95:5) to afford 24.7 g (91% yield) of the desired spiro compound as white crystals which were characterized as follows:

melting point 46° C.;

R_(f) (n-pentane/Et₂O, 95:5) 0.50;

[α]_(D) ^(rt) +17.4 (c=1.15, CHCl₃);

UV (MeOH) λ_(max) 255 nm;

IR (KBr): ν 3061, 2994, 2949, 2924, 2856, 1665, 1623, 1444, 1414, 1380, 1333, 1294, 1282, 1261, 1212, 1179, 1132, 1087, 1048, 1024, 986, 960, 926, 905, 813 and 733 cm⁻¹;

¹H NMR (500 MHz, CDCl₃): δ 12.1 (1 H, s), 5.68 (1 H, dt, J=10.1, 3.7 Hz), 5.45 (1 H, dt, J=10.1, 2.0 Hz), 3.76 (3 H, s), 2.32-2.22 (2 H, m), 2.16 (1 H, ABm, J=18.3 Hz), 2.12 (1 H, ABm, J=18.3 Hz), 2.00-1.94 (2 H, m), 1.73-1.50 (4 H, m) and 1.44-1.38 (2 H, m) ppm;

¹³C NMR (75 MHz, CDCl₃): δ 172.8 (C), 170.8 (C), 134.0 (CH), 127.1 (CH), 96.5 (CH), 51.4 (CH₃), 40.8 (CH₂), 34.1 (CH₂), 33.4 (CH₂), 25.4 (CH₂), 19.3 (CH₂) and 18.7 (CH₂) ppm;

MS m/z (%) 222 (M⁺, 4), 194 (14), 128 (15), 107 (14), 94 (68), 79 (100), 77 (36), 65 (20), 55 (36) and 41 (45); and

elemental analysis: calculated for C₁₃H₁₈O₃: C, 70.24; H, 8.16; found: C, 70.15; H, 8.18.

j) Preparation of spiro[5.51undec-7-en-2-one (10)

To a solution of the 2-hydroxy-spiro[5.5]undecan-2,7-diene-3-carboxylic acid methyl ester isomer (9) (19.0 g, 85.0 mmol) in DMSO (66 mL) was added NaCl (5.5 g, 93.5 mmol) and H₂O (4.6 mL, 255 mmol). The reaction mixture was heated at 160° C. for 6 hours then cooled and poured into H₂O (100 mL). The aqueous layer was extracted with Et₂O (5×) and the combined organic layers dried over anhydrous MgSO₄. The solvent was removed in vacuo and the resulting residue purified over silica (n-pentane/Et₂O, 95:5) to afford 13.3 g (95% yield) of the desired product as a colorless oil which was characterized as follows:

R_(f) (n-pentane/Et₂O, 9:1) 0.36;

[α]_(D) ^(rt) +68.9 (c=1.02, CHCl₃);

IR (KBr film): ν 3014, 2932, 2873, 1713, 1446, 1422, 1345, 1312, 1287, 1227, 1204, 1064, 1009, 923, 887, 730 and 529 cm⁻¹;

¹H NMR (500 MHz, CDCl₃) δ 5.64 (1 H, ABt, J=10.1, 3.7 Hz), 5.42 (1 H, AB(fs)), J=10.1 Hz), 2.32-2.26 (2 H, m), 2.21 (1 H, AB, J=13.9 Hz), 2.21 (1 H, AB, J=13.9 Hz), 1.95-1.82 (4 H, m), 1.72 (1 H, m), 1.63-1.52 (3 H, m) and 1.51-1.40 (2 H, m) ppm;

¹³C NMR (50 MHz, CDCl₃) δ 211.6 (C), 133.7 (CH), 126.9 (CH), 52.8 (CH₂), 40.9 (CH₂), 39.4 (C), 36.9 (CH₂), 33.4 (CH₂), 24.9 (CH2), 21.6 (CH₂) and 18.3 (CH₂) ppm;

MS m/z (%) 164 (M⁺, 61), 146 (8), 136 (11), 131 (17), 121 (39), 107 (89), 91 (41), 79 (100), 67 (17), 55 (18) and 42 (33); and

elemental analysis: calculated for C₁₁H₁₆O: C, 80.44; H, 9.82; found: C, 80.33; H, 9.95.

k) Preparation of 1,4-dioxa-dispiro[4.1.5.3]pentadec-8-ene (11)

To a solution of the spiro[5.5]undec-7-en-2-one isomer (10) (10 g, 61 mmol) in toluene (150 mL) was added HO(CH₂)₂OH (10 mL, 179 mmol) and p-toluenesulfonic acid.H₂O (0.57 g, 3 mmol). The reaction mixture was brought to reflux and the H₂O generated was removed by azeotropic distillation (using a Dean-Stark separator). The reaction mixture was then cooled and washed with a saturated NaHCO₃ solution. The aqueous phase was extracted with Et₂O and the combined organic layers dried over anhydrous Na₂SO₄. The solvent was removed in vacuo and the resulting residue purified over silica (isooctane/EtOAc, 95:5) to afford 12.1 g (95% yield) of the desired acetal which was characterized as follows:

R_(f) (isooctane/EtOAc, 95:5) 0.34;

[α]_(D) ^(rt) +25.2 (c=1.03, CHCl₃);

IR (KBr film) ν 2933, 2871, 2837, 1643, 1147, 1360, 1317, 1283, 1241, 1219, 1178, 1156, 1074, 1051, 1013, 947, 926, 888, 854, 821 and 732 cm⁻¹;

¹H NMR (500 MHz, CDCl₃): δ 5.67 (1 H, AB, J=10.2 Hz), 5.59 (1 H, ABt, J=10.2, 3.6 Hz), 3.94-3.89 (4 H, m), 1.96-1.92 (2 H, m), 1.68-1.50.(10 H, m) and 1.40-1.30 (2 H, m) ppm;

¹³C NMR (50 MHz, CDCl₃): δ 136.2 (CH), 125.6 (CH), 109.3 (C), 64.1 (CH₂), 63.9 (CH₂), 45.4 (CH₂), 37.3 (CH₂), 35.8 (C), 35.0 (CH₂), 25.5 (CH₂), 19.5 (CH₂) and 18.9 (CH₂) ppm;

MS m/z (%) 208 (M⁺, 12), 193 (14), 165.(70), 125 (17), 99 (100), 86 (62), 79 (38) and 41 (32).

Scheme II below outlines a sequence of eight process steps for the preparation of further intermediates and precursors, in particular intermediate 14b, containing suitable substituents of the F ring in order to obtain the final compound of the present invention. Each step of this scheme 11 will now be illustrated in details, based on specific starting compounds, reactive agents, catalysts, solvents, temperature ranges and the like, but the skilled person will understand that the specific materials and conditions disclosed herein may be replaced with similar or equivalent materials and conditions without significantly altering the resulting product of the relevant step, except maybe for the reaction yield of such step.

Scheme II

In a first step (a), the relevant 1,4-dioxa-dispiro[4.1.5.3]pentadec-8-ene stereoisomer (11) obtained in the eleventh and last step of scheme (I) is submitted to oxidative cleavage by any suitable method, for instance by ozonolysis at low temperature and optionally in the presence of a catalyst. More specifically, said step may be performed in two sub-steps, first including complex formation in the presence of ozone and a solvent, and secondly with use of trimethylphosphite as a reducing agent:

(preferred reagents and conditions for step (a) include: first (i) O₃, methanol as a solvent; temperature about −90° C. to −70° C., more specifically −78° C.; then (ii) P(OCH₃)₃; temperature ranging from about −78° C. to about −25° C.).

In a second step (b), the 7-(4-oxo-butyl)-1,4-dioxaspiro[4.5]decane-7-carbaldehyde stereoisomer resulting from step (a) is submitted to an aldol condensation preferably in the presence of a suitable solvent and preferably in the presence of an effective amount of a suitable basic catalyst such as, but not limited to, a triflate reagent:

(preferred reagents and conditions for step (b) include: (PhCH₂)₂NH₂ ⁺CF₃CO₂ ⁻ as a catalyst, THF as a solvent; temperature ranging from about 10° C. to about 40° C.).

In a third step (c), the 1,4-dioxa-dispiro[4.1.4.3]tetradec-8-ene-9-carbaldehyde stereoisomer resulting from step (b) is reduced in the presence of a suitable reducing agent such as, but not limited to, hydrogen and in the presence of a suitable solvent and optionally in the presence of an effective amount of a suitable catalyst such as platinum supported onto a carrier:

(preferred reagents and conditions for step (c) include: H₂ as a reducing agent, Pt/C as a catalyst; ethyl acetate as a solvent; temperature ranging from about 10° C. to about 40° C.).

In a fourth step (o), the (1,4-dioxa-dispiro[4.1.4.3]tetradec-9-yl) methanol stereoisomer 14b resulting from step (c) is activated for nucleophilic displacement, e.g. sulfonated, in the presence of a suitable sulfonating agent and a suitable solvent and optionally in the presence of an effective amount of a suitable catalyst:

(preferred reagents and conditions for step (o) include: toluenesulfonyl chloride or methanesulfonyl chloride as a sulfonating agent, dimethylaminopyridine (DMAP) as a catalyst, pyridine as a solvent; reaction temperature ranging from about 10° C. to about 40° C.).

In a fifth step (p), the toluene-4-sulfonic acid 1,4-dioxa-dispiro[4.1.4.3]tetradec-9-ylmethyl ester stereoisomer resulting from step (o) is submitted to a halo-de-sulfonyloxy-substitution somewhat similar to a Finkelstein reaction, preferably in the presence of a suitable solvent:

(preferred reagents and conditions for step (p) include: Nal, NaHCO₃, CH₃CN as a solvent; reflux temperature of the solvent; substitution may also be effected with a bromide or a chloride instead of an iodide).

In a sixth step (q), the 9-iodomethyl-1,4-dioxa-dispiro[4.1.4.3]tetradec-9-yl methyl ester resulting from step (p) is submitted to a 1,4-addition reaction by means of methyl acrylate, preferably in the presence of a solvent and optionally in the presence of an effective amount of a suitable catalyst:

(preferred reagents and conditions for step (q) include: CH₂═CHCO₂CH₂CH₃, Zn and NiCl₂.6H₂O as catalyst components, pyridine as a solvent; reaction temperature ranging from about 10° C. to about 40° C.).

In a seventh step (r), the 4-(1,4-dioxa-dispiro[4.1.4.3]tetradec-9-yl)-butyric acid ethyl ester resulting from step (q) is alkylated, e.g. by reaction with a suitable organometallic species, preferably a Grignard reagent, preferably in the presence of a suitable solvent:

(preferred reagents and conditions for step (r) include: CH₃MgBr as a Grignard reagent, THF as a solvent; temperature ranging from about 0° C. to about 40° C.).

In an eighth step (s), the 5-(1,4-dioxa-dispiro[4.1.4.3] tetradec-9-yl)-2-methyl pentanol resulting from step (r) is hydrolysed, preferably under acidic conditions and in the presence of an aqueous solvent:

(preferred reagents and conditions for step (s) include: p-toluenesulfonic acid as an acidic catalyst, acetone/water as a solvent medium; reaction temperature ranging from about 0° C. to about 40° C.).

EXAMPLE 2 Preparation of 2-(4-hydroxy-4-methylpentyl)-spiro[4.5]decan-7-one (19)

The preparation of 2-(4-hydroxy-4-methylpentyl)-spiro[4.5]decan-7-one was effected via the principles of the eight steps synthetic route described above, and more especially as outlined hereunder.

a) Preparation of 7-(4-oxo-butyl)-1,4-dioxaspiro[4.51decane-7-carbaldehyde (12)

Ozone gas was bubbled into a solution of 1,4-dioxa-dispiro[4.1.5.3]pentadec-8-ene (11) (3.0 g, 14.4 mmol) in MeOH (30 mL) at −78 ° C. Once the reaction was completed as determined by thin layer chromatography (TLC), argon gas was bubbled through the reaction solution for 10 minutes after which phosphorous acid trimethyl ester (3 mL, 25.4 mmol) was added. The temperature was allowed to rise to −40° C. and stirring was continued for an additional 30 minutes. The solvent was then removed under reduced pressure to afford the desired product which was used without further purification in the next step.

b) preparation of 1,4-dioxa-dispiro[4.1.4.31tetradec-8-ene-9-carbaldehyde (13)

To the 7-(4-oxo-butyl)-1,4-dioxaspiro[4.5]decane-7-carbaldehyde (12) obtained in the previous step, was added dibenzylammonium trifluoroacetate (0.4 M solution in THF; 12 mL, 4.8 mmol). The resulting mixture was stirred at room temperature for 3 hours after which the solvent was removed in vacuo. The resulting residue was purified over silica (cyclohexane/EtOAc, 8:2 mixture) to provide 2.3 g (72% yield) of the desired unsaturated aldehyde as a colorless oil which was characterized as follows:

R_(f) (isooctane/EtOAc, 7:3) 0.33;

optical rotation at room temperature: [α]_(D) ^(rt) +7.3 (c=0.89, CHCl₃);

UV (MeOH) λ_(max) 239 nm;

IR (KBr film) ν 3392, 2939, 2889, 1681, 1618, 1475, 1448, 1363, 1313, 1243, 1213, 1168, 1110, 1063, 1050, 948, 827 and 712 cm⁻¹;

¹H NMR (500 MHz, CDCl₃) δ 9.75 (1 H, s), 7.00 (1 H, s), 3.91-3.90 (4 H, m), 2.49-2.46 (2 H, m), 1.87 (1 H, ABt, J=13.2, 7.4 Hz), 1.81 (1 H. ABt, J=13.0, 7.0 Hz), 1.74-1.53 (7 H, m) and 1.40 (1 H, m) ppm;

¹³C NMR (75 MHz, CDCl₃) δ 190.9 (CH), 160.0 (CH), 145.1 (C), 108.7 (C), 64.3 (CH₂), 64.2 (CH₂), 51.1 (C), 44.2 (CH₂), 37.4 (CH₂), 35.8 (CH₂), 34.6 (CH₂), 26.5 (CH₂) and 20.9 (CH₂) ppm; and

MS m/z (%) 222 (M⁺, 9), 195 (19), 179 (25), 151 (9), 131 (6), 107 (11), 99 (100), 86 (70), 79 (31), 77 (34) and 55 (33).

c) Preparation of (1,4-dioxa-dispiro[4.1.4.3]tetradec-9-yl)methanol (14)

A mixture of 1,4-dioxa-dispiro[4.1.4.3]tetradec-8-ene-9-carbaldehyde (13) (500 mg, 2.5 mmol) and Pt (5 weight-% on activated carbon; 488 mg, 0.125 mmol) in EtOAc (10 mL) was placed under H₂ (1 atmosphere) and vigorously stirred at room temperature for 36 hours. The mixture was then filtered, through Celite and the filtrate was concentrated in vacuo to afford an oil which was purified over silica (eluent: isooctane/EtOAc mixtures, elution gradient from 7:3 to 1:1) to afford a 1:1 mixture of epimeric alcohols (450 mg, 88%) which were then separated by HPLC over silica (toluene/EtOAc, 8:2).

Characterization was as follows:

MS M/z(%) 226 (M⁺, <1), 195 (3), 183 (73), 165 (3), 121 (4), 113 (9), 99 (100), 86 (26), 79 (10), 67 (7), 55 (14) and 41 (12); and

elemental analysis: calculated for C₁₃H₂₂O₃: C, 68.99; H, 9.80; found: C, 68.81; H, 9.90.

d) Preparation of toluene-4-sulfonic acid 1.4-dioxa-dispiro[4.1.4.31tetradec-9-ylmethyl ester (15)

To a solution of (1,4-dioxa-dispiro[4.1.4.3]tetradec-9-yl)methanol (14) (1.20 g, 5.3 mmol) in dry pyridine (12 mL) at 0 ° C. was added 4-dimethylaminopyridine (catalytic amount) and p-toluenesulfonyl chloride (2.04 g, 10.6 mmole). The reaction mixture was stirred at room temperature for 24 hours and then poured into a 2 M HCl solution (50 mL). The aqueous solution was extracted with CH₂Cl₂and the combined organic layers were washed with a 2 M HCl solution and then with a saturated K₂CO₃ solution. After drying over anhydrous MgSO₄, the solvent was removed in vacuo to afford the desired product as a colorless oil which was used without further purification in the next step.

e) Preparation of 9-iodomethyl-1,4-dioxa-dispiro[4.1.4.31tetradec-9-ylmethyl ester (16)

Toluene-4-sulfonic acid 1,4-dioxa-dispiro[4.1.4.3]tetradec-9-ylmethyl ester (15) obtained in the previous step was dissolved in CH₃CN (20 mL). To this solution were added Nal (2.39 g, 15.9 mmole) and NaHCO₃ (catalytic amount) and the reaction mixture was then refluxed for 5 hours. The mixture was allowed to cool to room temperature and the solvent was removed in vacuo. The resulting residue was dissolved in CH₂Cl₂(40 mL) and the solution was washed with H₂O and dried over anhydrous MgSO₄. The solvent was removed in vacuo and the resulting residue was purified over silica (isooctane/EtOAc, 9:1) to afford 1.48 g (83% yield) of the desired iodide which was characterized as follows:

MS m/z: 293, 209, 99 (100 %), 86, 79, 55, and 41; and

elemental analysis: calculated for C₁₃H₂₁IO₂: C, 46.44; H, 6.30. Found: C, 46.27; H, 6.41

f) preparation of 4-(1,4-dioxa-dispiro[4.1.4.3]tetradec-9-yl)-butyric acid ethyl ester (17)

To a suspension of zinc powder (680 mg, 10.4 mmol) in dry pyridine (15 mL) was added CH₂═CHCO₂Et (1.13 mL, 10.4 mmol) followed by NiCl₂.6H₂O (593 mg, 2.5 mmol) and the reaction mixture was stirred at 65° C. for 2 hours. The mixture was allowed to cool to room temperature and 9-iodomethyl-1,4-dioxa-dispiro[4.1.4.3] tetradec-9-yl methyl ester (16) (700 mg, 2.08 mmol) dissolved dry pyridine (8 mL) was added dropwise and the reaction mixture stirred at room temperature for 3 hours. The mixture was poured into EtOAc (30 mL) and the resulting precipitate removed by filtration through a bed of Celite. The filtrate was collected and washed two times with a 1 M HCl solution, then saturated NaHCO₃, and dried over anhydrous MgSO₄. The solvent was removed in vacuo and the resulting residue was purified over silica (isooctane/EtOAc, 9:1) to afford 530 mg (82% yield) of the desired ester as a colorless oil which was characterized as follows:

MS m/z:310, 267, 195, 167, 151, 113, 99 (100%), 86, 55 and 41; and

elemental analysis: calculated for C₁₈H₃₀O₄: C, 69.64; H, 9.74; found: C, 69.80; H, 9.87.

g) preparation of 5-(1,4-dioxa-dispiro[4.1.4.3]tetradec-9-yl)-2-methyl-pentan-2-ol (18)

To a solution of 4-(1,4-dioxa-dispiro[4.1.4.3]tetradec-9-yl)-butyric acid ethyl ester (17) (530 mg, 1.71 mmol) in dry THF (25 mL) at 0° C. was added dropwise methyl-magnesium bromide (3 M solution in Et₂O; 2.83 mL, 8.5 mmol) and the reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by adding a saturated NH₄Cl solution (30 mL), the aqueous layer was then separated and repeatedly extracted with Et₂O. The combined organic layers were dried over anhydrous MgSO₄ and the solvents removed in vacuo. The resulting residue was purified over silica (isooctane/EtOAc, 8:2) to afford 451 mg (89 % yield) of the desired alcohol which was-characterized as follows:

optical rotation at room temperature: [α]_(D) ^(rt) −4.8 (c=0.85, CHCl₃);

IR (KBr film) ν 3438, 2932, 2868, 1445, 1362, 1317, 1279, 1173, 1100, 1056, 946, 908, 857 and 836 cm⁻¹;

¹H NMR (500 MHz, CDCl₃) δ 3.94-3.89 (4 H, m), 1.92-1.85 (2 H, m), 1.79-1.73 (1 H, m), 1.63-1.42 (11 H, m), 1.38-1.24 (6 H, m), 1.20 (6 H, s), 1.17-1.13 (1 H, m), 0.95-0.88 (1 H, m) ppm;

¹³C NMR (75 MHz, CDCl₃) δ 109.6 (C), 71.1 (C), 64.0 (CH₂), 45.9 (CH₂), 45.4 (CH₂), 44.2 (CH₂), 43.5 (C), 39.0 (CH₂), 38.6 (CH), 37.4 (CH₂), 35.1 (CH₂), 31.6 (CH₂), 29.3 (CH₃), 23.5 (CH₂), 21.2 (CH₂) ppm; and

MS m/z (%)281 (2), 253 (25), 195 (4), 167 (3), 151 (3), 113 (9), 99 (100), 86 (22), 55 (18) and 41 (12).

h) Preparation of 2-(4-hydroxy-4-methylpentyl)-spiro[4.5]decan-7-one (19)

To a solution of 5-(1,4-dioxa-dispiro[4.1.4.3]tetradec-9-yl)-2-methyl-pentan-2-ol, 18, (420 mg, 1.42 mmol) in acetone (9 mL) was added H₂O (catalytic amount) and p-toluenesulfonic acid (catalytic amount). The reaction mixture was stirred at room temperature overnight and then dried over anhydrous MgSO₄. The solution was filtered through silica and the filtrate concentrated under reduced pressure. The resulting residue was purified over silica (isooctane/EtOAc, 7:3) to afford 312 mg (86 % yield) of the desired ketone as a colorless oil which was characterized as follows:

[α]_(D) ^(rt) −3.1 (c=0.90, CHCl₃);

IR (KBr film) ν 3436, 2934, 2863, 1706, 1460, 1443, 1425, 1376, 1311, 1288, 1228, 1176, 1156, 1078, 1025, 937 and 909 cm⁻¹;

¹H NMR (500 MHz, CDCl₃) δ 2.30-2.24 (2 H, m), 2.23 (2 H, s), 1.96-1.79 (4 H, m), 1.68-1.64 (3 H, m), 1.53 (1 H, ddd, J=13.3, 9.1, 4.8 Hz), 1.44-1.40 (3 H, m), 1.35-1.25 (5 H, m), 1.22-1.16 (1 H, m), 1.19 (6 H, s), 0.97 (1 H, dd, J=12.6, 10.3 Hz) ppm;

¹³C NMR (75 MHz, CDCl₃) δ 212.1 (C), 70.9 (C), 53.7 (CH₂), 47.3 (C), 45.3 (CH₂), 44.1 (CH₂), 41.2 (CH₂), 38.8 (CH), 38.2 (CH₂), 38.1 (CH₂), 36.9 (CH₂), 31.4 (CH₂), 29.3 (CH₃), 23.8 (CH₂) and 23.4 (CH₂) ppm; and

MS m/z (%) 237 (7), 234 (3), 194 (6), 176 (12), 161 (9), 136 (18), 123 (10), 110 (18), 93 (31), 81 (16), 79 (16), 59 (100), 55 (31), 43 (30) and 41 (30).

Scheme III is exemplary of a procedure for coupling the substituted spiro[4.5]-decan-7-yl unit of the CF-ring scaffold with an A-ring scaffold (the synthesis of the A-ring scaffold being performed as known in the prior art). The following scheme includes a sequence of three steps for making the vitamin D₃ analog of this invention through a further set of intermediates. Each step will now be illustrated in details, based on specific starting compounds, reactive agents, catalysts, solvents, temperature ranges and the like, but the skilled person will readily understand that the specific materials, conditions and number of process steps disclosed herein may be replaced with similar or equivalent materials and conditions without significantly altering the resulting product of the relevant step or the final resulting product, except maybe for the yield thereof.

Scheme III

In a first step (t), the hydroxyl group of intermediate (19) is protected by means of a suitable 0-protecting group. Conventional O-protecting groups for protecting hydroxyls are well known in the art and include silyl, acyl, lower alkyl monocarbonyl, lower alkenyl monocarbonyl, alkoxycarbonyl, alkyl-carbonyl, lower alkoxycarbonyl-alkylcarbonyl, and arylcarbonyl groups. Exemplary O-protecting groups include, but are not limited to, alkoxycarbonyls (e.g. methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-isopropoxy-carbonyl, n-butyloxy-carbonyl, isobutyloxycarbonyl, sec-butyloxycarbonyl, t-butyloxycarbonyl, 2-ethylhexyloxycarbonyl, cyclohexyloxycarbonyl, methyloxy-carbonyl and the like), alkoxyalkoxycarbonyls (e.g. methoxymethoxycarbonyl, ethoxymethoxy-carbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl, 2-butoxyethoxy-carbonyl, 2-methoxyethoxymethoxycarbonyl and the like), haloalkoxy-carbonyls (e.g. 2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl, 2,2,2-trichlorethoxycarbonyl and the like), unsaturated alkoxycarbonyls (e.g., allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl, 3-methyl-2-butenoxycarbonyl and the like), substituted benzyloxycarbonyls (e.g. benzyloxycarbonyl, p-methylbenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl, 3,5-dimethylbenzyl-oxycarbonyl, p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl and the like) and substituted phenoxycarbonyls (e.g. phenoxycarbonyl, p-nitro-phenoxycarbonyl, o-nitrophenoxycarbonyl, 2,4-dinitrophenoxycarbonyl, p-methylphenoxycarbonyl, m-methylphenoxycarbonyl, o-bromophenoxycarbonyl, 3,5-dimethylphenoxycarbonyl, p-chlorophenoxycarbonyl, 2-chloro-4-nitrophenoxy-carbonyl and the like. A broader review of such groups may be found e.g. in Greene et al., Protective Groups in Organic Synthesis, 2^(nd) ed., John Wiley & Sons, Inc., New York, (1991).

Exemplary lower alkyl monocarbonyl groups which may be present in such O-protecting groups include, but are not limited to, acetyl, propionyl, butyryl, isobutyryl and the like. Exemplary lower alkenyl monocarbonyl groups which may be present in such are O-protecting groups include, but are not limited to, acryloxy, methacryloxy and the like. Exemplary lower alkoxycarbonyl-alkylcarbonyl groups include, but are not limited to, methoxycarbonyl-methylcarbonyl, ethoxycarbonyl-methylcarbonyl, ethoxy-carbonyl-ethylcarbonyl and the like. Exemplary arylcarbonyl groups include, but are not limited to, benzoyl, p-methoxybenzoyl, 3,4,5-trimethoxy benzoyl, p-chlorobenzoyl, 2,4-dichlorobenzoyl, 3,5-dichlorobenzoyl; diphenylacetyl, 1-naphthaleneacetyl, 2-naphthaleneacetyl and the like. Exemplary silyl groups may be represented by the formula —SiR′R″R′″, wherein each of R′, R″ and R′″ is independently selected from the group consisting of C₁₋₇ alkyl, C₂₋₇ alkenyl, C₃₋₁₀ cycloalkyl and aryl.

Conventional O-protecting groups, as set forth above, may be positioned during this first step by using standard procedures well known in the art.

(preferred reagents and conditions for step (t) include: trimethylsilylimidazole as a protecting group-containing reagent, THF as a solvent; reaction temperature ranging from about 10° C. to about 40° C.).

In a second step (u), the 2-(4-methyl-4-trimethylsilanyloxy-pentyl)-spiro [4.5]decan-7-one from step (t) is submitted to a Horner-Wittig reaction involving a phosphine oxide corresponding to the A-ring scaffold and wherein hydroxy groups are protected by conventional O-protecting groups (such as described herein-above), optionally in the presence of an effective amount of a suitable catalyst (preferably an organometallic species derived from an alkaline metal such as, but not limited to, an alkyllithium) and optionally in the presence of a suitable solvent such as an ether:

(preferred reagents and conditions for step (u) include: 5-[2-(diphenyl-phosphinoyl)-ethylidene]-bis-(tert-butyl-dimethylsilanyloxy)-cyclohexane as a phosphine oxide reagent, n-butyl lithium as a catalyst, THF as a solvent; temperature ranging from about −90° C. to about −60° C.). The 19-nor A-ring phosphine oxide was prepared as known in the prior art.

Then in a third and final step (v), oxygen deprotection is effected under standard conditions for this kind of reaction:

(preferred reagents and conditions for step (v) include: tetrabutylammonium fluoride as a reagent; THF as a solvent; reaction, temperature ranging from about 10° C. to about 40° C.).

EXAMPLE 3 Preparation of (1R,3R)-5-{2-[(2R,5R)-2-(4-hydroxy-4-methyl-pentyl)spiro[4.5]dec-(7E)-ylidene]-ethylidene}-cyclohexane-1,3-diol (23) a) Preparation of 2-(4-methyl-4-trimethylsilanyloxy-pentyl)-spiro(4.5]decan-7-one (20)

To a solution of 2-(4-hydroxy-4-methylpentyl)-spiro[4.5]decan-7-one (19) (50 mg, 0.198 mmole) in dry THF (1 mL) was added 1-(trimethylsilyl)imidazole (0.146 mL, 0.99 mmole). The reaction mixture was stirred at room temperature for 4 hours and the solvent was then removed in vacuo. The resulting residue was purified over silica (petroleum ether/EtOAc, 95:5) to afford the desired product which was used directly for the next reaction step.

b) Preparation of 7-{2-[3,5-bis-(tert-butyl-dimethyl-silanyloxy)-cyclohexylidene]-ethylidene}-2-(4-methyl-4-trimethylsilanyloxy-penty)-spiro[4.5]decane (22a/22b)

To a solution of 5-[2-(diphenyl-phosphinoyl)-ethylidene]-bis-(tert-butyl-dimethyl-silanyloxy)-cyclohexane (21) (which may be obtained according to the procedure disclosed by Perlman et al. in Tetrahedron Lett. (1991) 32, 7663-7666), (225 mg, 0.40 mmol) in dry THF (4 mL) at −78° C. was added dropwise n-BuLi (2.5 M solution in n-hexane; 0.167 mL, 0.417 mmol) and the reaction mixture was stirred at −78° C. for 1 hour. A solution of 2-(4-methyl-4-trimethylsilanyloxy-pentyl )-spiro[4.5]decan-7-one (20) obtained in the above step, in dry THF (3.5 mL) was added dropwise and the reaction mixture was stirred at −78° C. for 4 hours. The temperature was allowed to rise to room temperature and the solvent removed in vacuo. The resulting residue was purified over silica (petroleum ether/EtOAc, 95:5) to afford the desired product as a mixture of isomers which was used without further purification.

c) Preparation of (1R,3R)-5-{2-[(2S,5R)-2-(4-hydroxy-4-methyl-pentyl)-spiro[4.5]dec-(7E)-ylidene]-ethylidene}-cyclo-hexane-1.3-diol (23)

To the compound (22a) obtained in the above step, was added n-Bu₄NF (1 M solution in THF; 1.98 mL, 1.98 mmol), the reaction mixture was stirred at room temperature overnight. The isomer was formed in a 3:1 ratio. The reaction mixture was purified over silica (CH₂Cl₂/Me₂CO, 7:3) to afford (95% yield) of a mixture of isomers. The desired product was then separated from the corresponding 7Z-isomer by HPLC and characterized as follows:

R_(f) (CH₂Cl₂/Me₂CO, 6:4) 0.37;

UV (MeOH) λ_(max) 259, 249 and 241 nm;

IR (KBr film) ν 3360, 2930, 2864, 1612, 1443, 1361, 1218, 1049, 976, 936, 908, 862, 806, 733 cm⁻¹;

¹H NMR (500 MHz, CDCl₃) δ 6.25 (1 H, AB, J=11.3 Hz), 5.93 (1 H, AB, J=11.3 Hz), 4.09 (2 H, br s), 2.59 (1 H, dd, J=13.4, 3.5 Hz), 2.49 (1 H, dd, J=13.2, 3.7 Hz), 2.35 (1 H, dd, J=13.4, 7.2 Hz), 2.31-2.27 (1 H, m), 2.19-2.11 (2 H, m), 1.98 (2 H, s), 1.90-1.65 (6 H, m), 1.58-1.25 (14 H, m), 1.20 (3 H, s), 1.19 (3 H, s), 1.19-1.11 (1 H, m), 0.79 (1 H, dd, J=12.4, 10.4 Hz) ppm;

¹³C NMR (75 MHz, CDCl₃) δ 141.9 (C), 131.4 (C), 123.9 (CH), 118.2 (CH), 71.1 (C), 67.5 (CH), 67.2 (CH), 49.6 (CH₂), 45.2 (C), 45.0 (CH₂), 44.9 (CH₂), 44.1 (CH₂), 42.2 (CH₂), 39.9 (CH₂), 38.7 (CH), 37.9 (CH₂), 36.9 (CH₂), 36.8 (CH₂), 31.7 (CH₂), 29.3 (CH₃), 29.1 (CH₃), 28.7 (CH₂), 24.6 (CH₂), 23.4 (CH₂) ppm;

MS m/z (%) 358 (M⁺-H₂O, 22), 340 (13), 325 (5), 217 (9), 177 (36), 145 (32), 133 (23), 119 (30), 105 (39), 93 (77), 91 (67), 79 (60), 67 (56), 59 (100), 55 (54), 43 (80).

Medical Uses

The vitamin D₃ analog described herein has been found in many instances to exhibit useful biological activity (such as in one or more assays which are referenced herein) at a level below about 1 mM, more specifically below 1 μM. Consequently this compound is useful as an active ingredient in pharmaceutical formulations and more particularly useful in the manufacture of medicaments for the prevention and/or treatment of one or more of the disorders and diseases, especially bone disorders. In particular, (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro-[4.5]dec-7-ylidene]-ethyli-dene}-cyclohexane-1,3-diol is suitable for use in diseases and conditions which are related to calcium and phosphate metabolism such as, but not limited to, osteoporosis, renal osteodystrophy, Paget's disease, osteomalacia or disorders of the parathyroid function.

The compound described herein also has a selective activity on cell function. One key selective action relates to the inhibition of cell proliferation. Non-limiting examples of cells which are a target of cell proliferation inhibition include both non-malignant cells, inter alia, keratinocytes and malignant cells, inter alia, breast carcinoma cells, leukemia cells. A second selective activity on cell function relates to induction of cell differentiation, for example, leukemia cells.

Diseases that can be prevented or treated with the compound of this invention therefore also include the following:

A) Immune Disorders:

i) For the purposes of the present invention, type (i) immune disorders are auto-immune diseases, inter alia, diabetes mellitus type 1, multiple sclerosis, lupus and lupus like disorders, asthma, glomerulonephritis, and auto-immune throiditis.

ii) For the purposes of the present invention, type (ii) immune disorders are selective dysfunctions of the immune system, inter alia, Acquired Immune Deficiency Syndrome (AIDS).

iii) For the purposes of the present invention, type (iii) immune disorders are medically induced immune disorders, inter alia, the body's rejection of foreign tissue due to tissue grafts (e.g. kidney, heart, bone marrow, liver, islets or whole pancreas, and skin).

iv) For the purposes of the present invention, type (iv) immune disorders are autoimmune and other inflammatory diseases, inter alia, rheumatoid arthritis.

v) For the purposes of the present invention, type (v) immune disorders are skin disorders. These disorders can be due to any immune system imbalance, inter alia, those characterized by hyperproliferation, inflammation, (auto)immune reactions. Non-limiting examples include psoriasis, dyskeratosis, and acne.

Because of the advantages exhibited by the compounds of the present invention, inter alia, a lower toxic effect on calcium and bone homeostasis, the present invention also relates to the use of the compounds described herein in combination with one or more other immuno-modulating or-immuno-suppressing drugs known to affect the immune system, inter alia cyclosporin A, tacrolimus (FK 506), rapamycin, leflunomide, mofetil, methylxanthine derivatives, glucocorticoids, monoclonal antibodies, cytokines and growth factors, for the manufacture of pharmaceutical preparations for the treatment or prevention of the above immune disorders.

B) Cell Proliferation Disorders:

The compound of the present invention is also suitable for use in the treatment or prevention of diseases, disease states, and-disorders which are due to abnormal cell proliferation in humans. Among these conditions are breast cancer, leukemia, myelo-dysplastic syndromes and lymphomas, squamous cell carcinomas and gastrointestinal cancers, melanomas, and osteosarcoma.

Another advantage related to the cell differentiation capacity of the compound of the present invention relates to the treatment or prevention of alopecia. The present invention therefore relates to methods of treating or preventing alopecia, especially when induced by chemotherapy or irradiation, in humans.

Each of the disease states or conditions which may be desirable to treat or prevent according to this invention may require differing levels or amounts of the compound described herein in order to obtain a therapeutic level. The formulator can determine this therapeutic amount by any of the known testing procedures known to the artisan. It is well known that the effective amount of a certain compound may be predicated on various parameters which are germane to delivery of an effective active ingredient, inter alia, in vivo activity, bioavailability, metabolism, ease of formulation, stability of compound formulation and the like.

Formulations

The present invention relates to compositions or formulations which comprise the compounds according to the present invention. In general, the compositions of the present invention comprise (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol in an amount effective for controlling bone disorders such as osteoporosis. The compositions of the present invention preferably comprise:

-   -   a)         (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylhexyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol         in an amount effective for controlling osteoporosis; and     -   b) one or more excipients.

In a particular aspect, the present invention relates to pharmaceutical compositions or formulations which comprise the compounds according to the present invention. In general, the compositions of the present invention comprise (1R,3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol in a therapeutically effective amount, preferably an amount effective for controlling osteoporosis. The pharmaceutical compositions of the present invention preferably comprise:

-   -   a)         (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylhexyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol         in an amount effective for controlling osteoporosis; and     -   b) one or more pharmaceutically acceptable excipients.

For the purposes of the present invention the terms “excipient” and “carrier” are used interchangeably throughout the description of the present invention and said terms are defined herein as ingredients which are used in the practice of formulating a safe and effective pharmaceutical composition.

The formulator will understand that excipients, especially pharmaceutically acceptable excipients are used primarily to serve in delivering a safe, stable, and functional pharmaceutical composition or formulation, serving not only as part of the overall vehicle for delivery but also as a means for achieving effective absorption by the recipient of the active ingredient. An excipient may merely be an inert filler, or an excipient as used herein may be part of a pH stabilizing system or a coating to insure delivery of the ingredients safely to the desired part of the human body, e.g. the stomach. The formulator can also take advantage of the fact the vitamin D₃ compound of the present invention has improved cellular potency and that its pharmacokinetic properties as well as the oral bioavailability could be improved.

For the purpose of the present invention the term “effective amount”, as it-relates to the amount of (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol delivered to a patient in need of a treatment, is defined herein as an amount of (1R,3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol which produces the alleviation of symptoms, or the suppression of, a bone disorder such as but not limited to osteoporosis as may be measured directly, for example by a laboratory test or procedure which provides the quantitative level of this active ingredient in plasma, or indirectly, for example by the ability of the patient to no longer experience undesirable disease or disease state symptoms. Said symptoms are necessarily dependent upon one or more factors, inter alia age of the patient, degree of disease involvement, other diseases or disease states being simultaneously present, and therefore the effective amount will depend upon said factors and upon the desired therapeutical outcome (e.g. complete cure as in a chronic illness or temporary relief as in an acute illness condition). It is recognized that the compositions of the present invention can be delivered in various dosages and therefore, the effective amount can be determined on a patient by patient basis if necessary.

The amount of (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol necessary to achieve the “effective amount” or “therapeutic amount” as used herein, may typically be determined because of a range of factors. For the purpose of the present invention a first aspect of a “therapeutically effective amount” relates to pharmaceutical compositions which deliver a the vitamin D₃ compound according to the present invention in a manner wherein the plasma level of said compound is from about 1 μg/mL to about 100 mg/mL in humans or higher mammals. In addition, a further embodiment of an “effective amount” or “therapeutic amount” can also achieve a particular blood plasma level of (1R,3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol or a metabolite thereof. As it relates to the metabolite of the vitamin D₃ compound according to the present invention, this species may be biologically active, or may be a product of metabolism which is convenient to measure and/or is easily associated with the effective level of (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol which was administered.

A first aspect, the compositions of the present invention, more in particular the pharmaceutical compositions, comprise:

-   -   a) from about 1 pg to about 1000 mg of         (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol;         and     -   b) one or more excipients, more in particular pharmaceutically         acceptable excipients.

A second aspect relates to compositions, more in particular pharmaceutical compositions which achieve a blood plasma level of (1R,3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol or a metabolite thereof, wherein said plasma level is from about 0.001 pg/mL, yet more in particular from about 1 pg/mL to about 25 mg/mL in humans or higher mammals.

Administration of the compositions, more in particular pharmaceutical compositions of the present invention can achieve the desired therapeutic amounts in vivo as measured by the plasma level in various ways. It is not necessary to provide the therapeutic amount of compound in a single dose, for example a single pill or tablet. Therefore, the formulator can vary the size of the dosage, and therefore the amount of the active vitamin D₃ compound in the compositions of this invention. Non-limiting examples of compositions, more in particular pharmaceutical compositions according to the present invention, the effectiveness of which can be monitored by measuring plasma levels in mammals, more in particular in the human body, include:

-   -   a) from about 1 pg to about 1000 mg of         (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol;         and     -   b) one or more excipients, more in particular pharmaceutically         acceptable excipients.

Another embodiment of this aspect according to the present invention relates to the following compositions, more in particular pharmaceutical compositions comprising:

-   -   a) from about 1 ng to about 500 mg of         (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol;         and     -   b) one or more excipients, more in particular pharmaceutically         acceptable excipients.

Another embodiment of this aspect according to the present invention relates to the following compositions, more in particular pharmaceutical compositions comprising:

-   -   a) from about 1 mg to about 500 mg of         (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol;         and     -   b) one or more excipients, more in particular pharmaceutically         acceptable excipients.

A further embodiment of this aspect according to the present invention relates to the following compositions, more in particular pharmaceutical compositions comprising:

-   -   a) from about 100 mg to about 500 mg of         (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol;         and     -   b) one or more excipients suc as pharmaceutically acceptable         excipients.

In addition, the pharmaceutical compositions of the present invention can be administered to humans as frequently as necessary to achieve a therapeutic amount.

Method of Use

The present invention also relates to a method for controlling bone disorders such as osteoporosis in humans and mammals, especially higher mammal. More in particular, the present invention relates to a method for preventing or treating osteoporosis in humans and mammals, especially higher mammal. The present method comprises the step of administering to a human or higher mammal an effective amount of a composition, more in particular a pharmaceutical composition comprising (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol.

Because (1R, 3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro [4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol can be delivered in a manner wherein more than one site of biological control can be achieved, more than one disease state can be effectively treated at the same time by means of this compound. For example, calcium and phosphate metabolism may be controlled simultaneously, thus resulting in the capacity of treating one or more of the following diseases: plasma and bone mineral homeostasis, inter alia, osteomalacia, osteoporosis, renal osteodystrophy, and disorders of the parathyroid function.

Procedures

For the purposes of testing (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl )-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol as a therapeutic agent against osteoporosis and other calcium related conditions, 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], a Vitamin D metabolite, is used as the control.

Binding Affinity Evaluation

Affinity for Vitamin D Receptor (VDR)

The compound of the present invention has been evaluated for its affinity to bind to the Vitamin D receptor (VDR) versus the active metabolite 1α,25(OH)₂D₃. The methods employed and described herein have been used for determining steroid hormone and steroid hormone mimetic binding. Utilizing the tritium labeled analog, [³H]1α,25(OH)₂D₃, the compound of this invention has been evaluated for its binding to a high speed supernatant from porcine intestinal mucosa homogenates.

Incubation was performed at 4° C. for 20 hours and phase separation was obtained by adding dextran-coated charcoal. The relative affinity of (1R, 3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol was then calculated by determining the concentration needed to displace 50% of the [³H]1α,25(OH)₂D₃ from the VDR as compared to the amount of [³ H]1α,25(OH)₂D₃ displaced by non-tritium labeled 1α,25(OH)₂D₃, which was assigned the relative value of 100%.

Affinity for Human Vitamin D Binding Protein (hDBP)

(1R, 3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol was also evaluated for its affinity to bind to a Human Vitamin D Binding Protein (hDBP) versus the active metabolite 1α,25(OH)₂D₃. The methods employed and described herein have been used for determining steroid hormone and steroid hormone mimetic binding. Utilizing the tritium labeled analog, [³H]1α,25(OH)₂D₃, (1R,3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol was evaluated for its binding to a high speed supernatant from porcine intestinal mucosa homogenates.

An 5 μL solution of [³H]1α,25(OH)₂D₃ together with (1R,3R)-5-{(E)-(2R, 5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethyli-dene}-cyclohexane-1,3-diol, or with 1α,25(OH)₂D₃ in the case of the control experiment, dissolved in ethanol were added to glass tubes and incubated with hDBP (0.18 μM) made up to a final volume of 1 mL with a solution containing 0.01M Tris-HCl buffer and 0.154 M NaCl at pH 7.4. The solution was incubated for 3 hours at 4° C. Final phase separation was obtained by adding 0.5 mL of cold dextran-coated charcoal. The relative affinity (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol was then calculated by determining the concentration needed to displace 50% of the [³H]1α,25(OH)₂D₃ from hDBP as compared to the amount of [³H]1α,25(OH)₂D₃ displaced by non-tritium labeled 1α,25(OH)₂D₃, which was assigned the relative value of 100%.

Table I herein below lists the relative percentage of VDR binding and the relative percentage of hDBP binding of (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol versus control 1α,25(OH)₂D₃. TABLE I % % VDR hDBP Compound binding binding 1α,25(OH)₂D₃ (control) 100 100 (1R,3R)-5-{2-[(2R,5R-2-(4-hydroxy-4-methylpentyl)- 10 3 spiro[4.5]dec-(7E)-ylidene]-ethylidene}-cyclo-hexane- 1,3-diol Measurement of Cell Proliferation and Differentiation Breast Carcinoma Cells: MCF-7

The compound of the present invention was evaluated for its effect on cell proliferation. Malignant MCF-7 cells were cultured in DMEM nutrient mix F12 (HAM) medium which was supplemented with 10% heat inactivated FCS, glutamine (2 mM), penicillin (100 units/mL) and streptomycin (0.1 mg/mL). The cultures were maintained at 37° C. in an atmosphere of humidified air containing 5% CO₂. The MCF-7 cells were seeded at approximately 5×10³ cells/well in the DMEM modified medium in a 96-well microtiter plate. Each plate had wells made up to a final volume of 0.2 mL. After. 24 hours incubation, the control 1α,25(OH)₂D₃ or the compound to be tested was added in the appropriate concentration for an incubation period of 72 hours. Finally, 1 μCi of [³H]thymidine was added to each well and the cells were harvested after a further 4 hour incubation period. The cells were harvested with a Packard Harvester and the cell count measured by a Packard Topcount System. Results are expressed as percentage activity (at 50% of the dose response) in comparison with 1α,25(OH)₂D₃ as a control (having 100% activity)

Promyelocytic Leukemia Cells (HL-60)

The vitamin D analog of the present invention was evaluated for its effect on cell differentiation. Falcon tissue chambers were seeded with 4×10⁴ cells/cm of HL-60 cells using RPMI 1640 medium supplemented with 20e % FCS and gentamycin (50 μg/mL),in a final cell volume of 5 mL. The cultures were maintained at 37° C. in an atmosphere of humidified air containing 5% CO₂. After 24 hours incubation, the control 1αa,25(OH)₂D₃ and the compound to be tested were each dissolved in ethanol and added to the cell culture with the final concentration being less than 0.2%. After 4 days, the dishes were shaken to lose adherent cells. The cells were then washed twice in RPMI medium, counted, and then assayed for differentiation markers (NBT reduction assay). Superoxide production was measured as NBT reducing activity as described by Ostrem et al. in Proc. Natl. Acad. Sci. USA, (1987) 84: 2610-2614. HL-60 cells at 1×10⁶/mL were mixed with an equal volume of freshly prepared solution of phorbol 12-myristate 13-acetate (200 ng/mL) and NBT (2 μg/mL) and incubated for 30 minutes at 37° C. The percentage of cells containing black formazan deposits was determined using a hemacytometer. Results are expressed as percentage activity (at 50% of the dose response) in comparison with 1α,25(OH)₂D₃ as a control (having 100% activity)

Table II below provides both cell proliferation and cell differentiation test results for the compound according to the present invention. TABLE II Compound HL-60 MCF-7 1α,25(OH)₂D₃ (control) 100 100 (1R,3R)-5-{(E)—(2R,5R)-2-[2-(4-hydroxy-4- 200 450 methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}- cyclohexane-1,3-diol In Vivo Determination of Calcium Levels

Eight week old, male NMRI mice were obtained from the Animal Center of Leuven (Belgium) and fed a vitamin D-replete diet (0.2% calcium, 1% phosphate, 2000 U vitamin D/kg; Hope Farms, Woerden, The Netherlands). Groups of six mice were intra-peritoneously injected daily during 7 consecutive days with different doses of either 1α,25(OH)₂D₃ (0.1, 0.2 and 0.4 μg/kg/day) or (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro [4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol (0.4, 5 or 10 μg/kg/day). The control group was injected with vehicle (arachis oil). The average weight of each group of 6 mice was determined at the beginning and at the end of the experiment and are shown in Table III hereunder. The following parameters were evaluated: serum calcium and femur calcium. Serum calcium was measured by a microcolorimetric assay (Sigma, St. Louis, Mo.). Femurs were removed and femur calcium content was measured in HCl-dissolved bone ash (obtained by heating for 24 hours in an oven at 100° C.), using the same technique as for serum calcium.

(1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylide-ne]-ethylidene}-cyclohexane-1,3-diol afforded an estimated serum calcium level of 0.5%, as compared with 1α,25(OH)₂D₃ (assigned a value of 100%). The compound of the invention is therefore approximately 200 times less calcemic than 1α,25(OH)₂D₃.

Table III below presents average values of the serum and femur calcium levels in mice after intra-peritoneal injections of the relevant compound after 7 consecutive days. TABLE III Body Dose Serum Ca Femur Ca Weight Compound μg/kg/day mg/dl mg g Vehicle — 11.4 10.9 31.3 1α,25(OH)2D3 0.1 13.3 10.9 30.5 1α,25(OH)2D3 0.2 14.1 10.3 27.8 1α,25(OH)2D3 0.4 18.2 8.9 23.2 Test compound* 0.4 11.7 10.1 34.4 Test compound* 5 11.1 11.3 32.9 Test compound* 10 11.3 12.3 34.0 *(1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro-[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol Measurement of Prevention of Osteoporosis A) Primary Prevention of Osteoporosis by the Compound of the Present Invention.

12 Week old C3H female mice are subjected to bilateral ovariectomy or sham surgery. The animals are treated with a test compound or vehicle by oral gavage or intraperitoneally. Dosing is started 3 days after surgery and continued for 8-9 weeks.

Prior to the first treatment, in vivo measurements are performed to determine bone mineral density (BMD), bone mineral content (BMC) of total body and spine by dual-energy X-ray absorptiometry (DXA). Urine and serum is collected to measure calcium levels together with collagen cross-links in urine, and osteocalcin in serum. The animals are weighed regularly during the experimental period. After 4 weeks treatment urine and serum is again collected and biochemical parameters are determined. At the end of the experiment (8-9 weeks) urine is collected and DXA measurement is performed in vivo in order to determine BMD and BMC. After sacrificing the animals, the tibiae and femora are dissected and the following biochemical parameters are investigated: serum calcium, serum osteocalcin, urine calcium, urine collagen cross-links, femur calcium.

The tibiae are used for histomorphometric analysis and femurs for measurement of cortical and trabecular volumetric density and geometry by peripheral quantitative computed tomography (pQCT) ex vivo.

B) Secondary Prevention of Osteoporosis by the Compound of the Present Invention.

12 Week old C3H female mice are subjected to bilateral ovariectomy or sham surgery. The animals are treated with the vitamin D₃ analog of this invention or, as a control, with vehicle (arachis oil) by-oral gavage or intra-peritonally. Dosing is started 4 weeks after surgery and continued for 4 weeks.

Prior to the first treatment in vivo measurements are performed to determine bone mineral density (BMD), bone mineral content (BMC) of total body and spine by dual-energy X-ray absorptiometry (DXA). Urine and serum are collected in order to measure calcium levels together with collagen cross-links in urine and osteocalcin in serum. The animals can be weighed regularly during the experimental period. After 4 weeks treatment, urine and serum is again collected and biochemical parameters are determined and DXA measurement is performed in vivo to determine BMD and BMC. After sacrificing the animals, tibiae and femora are dissected. The following biochemical parameters are investigated: serum calcium, serum osteocalcin, urine calcium, urine collagen cross-links, and femur calcium.

The tibiae are used for histomorphometric analysis and femurs for measurement of cortical and trabecular volumetric density and geometry by peripheral quantitative computed tomography (pQCT) ex vivo.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

C) Efficacy of the Compound of the Present Invention in Three-Month Treatment

Efficacy was tested in 6 month old Sprague-Daley rats which were sham-operated or ovariectomized (OVX). Ovariectomized animals were sacrificed after two months as a pre-treatment control group, with remaining animals being treated for three months with vehicle or with (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol (at dosages of 0.3, 1,3 or 10 μg/kg/day respectively) by oral administration. At the end of treatment, blood was obtained by cardiac puncture for evaluation of serum calcium levels. Hypercalcemia was identified as a mean value 10% greater than the OVX-vehicle group. Bone efficacy was measured using micro-CT (Scanco μCT 40) evaluation of the vertebral cancellous bone volume compared to either the final OVX-group and/or the pre-treatment OVX group. The compound of this invention, (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol, was thus shown to be able to significantly increase vertebral bone volume above pre-treatment and final OVX groups at doses that did not induce hypercalcemia, as shown in FIG. 1.

The following publications are included herein by reference.

-   1. Hara, R. et al., J. Am. Chem. Soc. (1999), 121, 3072-3082. -   2. Hara, Y. et al. J. Org. Chem. (1975), 40, 2786-2791. -   3. De Buyck, L. et al. Bull. Soc. Chim. Belg. (1992), 101, 807-815. -   4. Perlman, K. L. et al. Tetrahedron Lett. (1991), 32, 7663-7666. -   5. Wali R. K et al., “Vitamin D receptor is not required for the     rapid actions of 1,25-dihydroxyvitamin D3 to increase intracellular     calcium and activate protein kinase C in mouse osteoblasts.” J Cell     Biochem 2003, 88: 794-801. -   6 Buitrago C. et al. “The vitamin D receptor mediates rapid changes     in muscle protein tyrosine phosphorylation induced by     1,25(OH)(2)D(3). Biochem Biophys Res Commun 2001, 289:1150-1156. -   7. Norman A. W. et al., “Molecular tools for study of genomic and     rapid signal transduction responses initiated by 1     alpha,25(OH)(2)-vitamin D(3).” Steroids 2002, 67: 457-466. -   8. Bouillon R. et al., “Structure-function relationships in the     vitamin D endocrine system.” Endocr Rev 1995,16, 200-257. -   9. Bouillon R. et al. Clin Chem 38:2055-60, (1992). 

1. The compound having the following structural formula:

i.e. (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol.
 2. A composition comprising the compound having the formula

i.e. (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-Hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol.
 3. The composition of claim-2, comprising: a) (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylhexyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol in an amount effective for controlling osteoporosis; and b) one or more excipients.
 4. The composition of claim 2, comprising: a) from about 1 pg to about 1000 mg of (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylhexyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol; and b) one or more excipients.
 5. The composition of claim 2, wherein said composition is a pharmaceutical composition.
 6. The composition of claim 2, wherein said composition is a pharmaceutical composition comprising a therapeutically effective amount of (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol.
 7. The composition of claim 2, wherein said composition is a pharmaceutical composition comprising a) a therapeutically effective amount of (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol and; b) one or more pharmaceutically acceptable excipients.
 8. The composition of claim 2, wherein said composition is a pharmaceutical composition comprising a) from about 1 pg to about 1000 mg of (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol; and b) one or more pharmaceutically acceptable excipients.
 9. A method of treating or preventing a bone disorder comprising administering to a human or a mammal an effective amount of a composition comprising the compound having the structural formula

i.e. (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol.
 10. The method of claim 9, wherein said composition further comprises one or more excipients.
 11. The method of claim 9, wherein said composition further comprises one or more pharmaceutically acceptable excipients.
 12. The method of claim 9, wherein -said bone disorder is osteoporosis.
 13. The method of claim 9, wherein said composition comprises: a) from about 0.001 mg to about 1000 mg of (1R,3R)-5-{(E)-(2R,5R)-2-[2-(4-hydroxy-4-methylpentyl)-spiro[4.5]dec-7-ylidene]-ethylidene}-cyclohexane-1,3-diol; and b) one or more excipients.
 14. The compound of claim 1, being characterized by the following chemical shifts of the proton nuclear magnetic resonance spectrum (500 MHz, CDCl₃): 6.23 (1 H, AB, J=11.3 Hz), 5.93 (1 H, AB, J=11.3 Hz), 4.11-4.02 (2 H, m), 2.68 (1 H, dd, J=13.2, 3.8 Hz), 2.47 (1 H, dd, J=13.3, 3.6 Hz), 2.28-2.16 (4 H, m), 2.00 (2 H, s), 1.93-1.71 (5 H, m), 1.57 (1 H, dd, J=12.9, 7.9 Hz), 1.53-1.43 (8 H, m), 1.36-1.26 (6 H, m), 1.21 (6 H, s), 1.21-1.13 (1 H, m), and 0.94 (1 H, dd, J=12.9, 9.4 Hz) ppm.
 15. The compound of claim 1, being characterized by the following chemical shifts of the carbon nuclear magnetic resonance spectrum (75 MHz, CDCl₃): 142.0, 131.4, 123.8, 117.9, 71.2, 67.5, 67.1, 50.7, 45.1, 44.8, 44.7, 44.2, 42.2, 39.4, 39.0, 38.2, 37.2, 31.8, 29.3, 29.2, 28.7, 24.4 and 23.4 ppm.
 16. The compound according to claim 1, being characterized by the following spectral absorption bands: i) UV (MeOH) λ_(max) 259, 249 and 241 nm; and ii) IR (KBr film) v 3364, 2930, 2857, 1617, 1444, 1364, 1212, 1151, 1049, 976, 938, 908, 864, 812 and 733 cm⁻¹.
 17. The compound according to claim 1, being characterized by the following mass spectrum data: m/z (%) 376, 358, 340, 325, 261, 243, 217, 177, 163, 145, 133, 119, 105, 93, 91, 79, 67, 59 (100%), 55, 43 and
 41. 